Signal enhancement of bispecific antibody-polymer probe for immunoassay use

ABSTRACT

An immunoassay method including reacting a sample from a patient with a bispecific antibody, wherein the bispecific antibody includes one antibody specific for a compound to be detected and a second antibody specific for a compound foreign to said patient sample, and subsequently reacting the patient sample with a polymer probe, wherein the polymer probe includes a compound recognized by the second antibody in the bispecific antibody complex and further includes at least two detectable signals; the bispecific antibody; and the polymer probe of the immunoassay method are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR DEVELOPMENT BACKGROUND OF THE INVENTION

[0001] An immunoassay utilizes antibodies to detect a compound ofchoice. However, the sensitivity of this detection is generally limitedby the amount of signal that can be carried either on the antibody, fora direct binding assay, or on the probe compound, in a competitiveinhibition assay. For example, in existing immunoassays, such asradioimmunoassay, ELISA, immunofluorescent assays orimmunochemiluminescent assays, too many signal entities, such asradioisotopes, horse radish peroxidase or alkaline phosphatase, attachedto the detection moieties invariably inactivate the antibody or denaturethe antigen and change the property of the detection probe. Therefore,in order to obtain more signal, additional antibody or probe must beadded. This, in turn, reduces the sensitivity of the assay, thecapability of the assay to detect minute quantities of the compound inquestion.

[0002] For all existing immunoassays, there is lag time for the compoundof interest to reach a high enough concentration in the serum to becomedetectable for diagnostic purposes. In the case of heart attacks, thereis a delay of 4-6 hours from the onset of chest pain until thediagnostic detection of CKMB, Troponin-T or I is possible. Myoglobin isdetectable earlier, but its specificity is low. If there were an assaythat could detect very minute increases of these indicator compounds inthe blood at an earlier point in time, then therapeutic interventioncould be started earlier and thereby bring about greater myocardialsalvage. In the case of cancer detection, where, e.g., tumor associatedantigens related to breast cancer or colon cancer, etc., are detected,treatment might be more effective if minute elevations of these antigenscould be detected at an early stage. Therefore, there is a need toincrease the sensitivity of the assay without adversely affecting thespecificity of the assay system.

SUMMARY OF THE INVENTION

[0003] The invention is directed to a method to increase the sensitivityof an immunoassay, by at least 10,000 fold, without losing specificity.This improvement is achieved by the use of a bispecific antibody complexand a unique detection signal probe capable of recognizing thebispecific antibody complex.

[0004] In one aspect, the invention features an immunoassay methodincluding reacting a sample from a patient with a bispecific antibody,wherein the bispecific antibody includes one antibody specific for acompound to be detected and a second antibody specific for a compoundforeign to said patient sample, and subsequently reacting the patientsample with a polymer probe, wherein the polymer probe includes acompound recognized by the second antibody in the bispecific antibodycomplex and further includes at least two detectable signals. Theinvention also features the bispecific antibody and the polymer probe ofthe method of the invention. Preferably, the sample from the patient isa blood or serum sample; the bispecific antibody includes an antimyosinantibody and an antibody against DTPA; and the polymer probe is apolylysine polymer and includes DTPA and at least six HRP as thedetectable signal compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof andfrom the claims, taken in conjunction with the accompanying drawings, inwhich:

[0006]FIG. 1a shows a standard ELISA according to the prior art;

[0007]FIG. 1b shows an immunoassay according to the invention; and

[0008]FIG. 2 is a graph showing competitive inhibition curves usingstandard ELISA (R11D10), bispecific antibody complex with standardsecondary antibody for signal production (BiMAb (Ab-HRP)), and themethod according to the invention (BiMAb(PL-DTPA-HRP)).

DETAILED DESCRIPTION OF THE INVENTION

[0009] The invention is directed to the development of a new approach tothe use of bispecific antibodies in immunoassays. The new specificantibody comprises one antibody specific for the compound associatedwith the pathological state to be detected and another antibody to achemical or reporter compound that is not found naturally in man. Thesetwo are chemically or genetically linked. The bispecific antibodycomplex constitutes the first line of interaction with the compound oneis attempting to detect. Normally many antibodies must react with thecompound to enable development of sufficient signal intensity fordetection. However, in the method of the invention, a novel detectionprobe is used, made up of any type polymer, such as polylysine or otherpolyamino acid, that is amenable to attachment of signal reagents andreporter compounds. The amount of signal reagent that can be used in agiven assay is limited only by the size of the polymer. Only a fewmolecules of the detection probe are therefore needed to provide thissignal. The signal probe is extremely versatile as any type of signalproducing compound such as radioactivity, chemical color producingenzymes or fluorescent probes can be attached to the polymer backbone.Signal amplification is not limited by the nature of the bispecificantibody complex itself.

[0010] Therefore, the immunoassay sensitivity can be amplified by atleast 10,000-fold compared to conventional immunoassays orimmunosandwich assays. Since early detection of many pathologicalstates, such as acute myocardial infarction and cancer, is limited bythe sensitivity of immunoassays to detect minute elevations of thepathologically associated compounds, an method and compounds of theinvention will enable diagnosis of disease states at a much earlier timethan previous assays, which may allow for better therapeuticintervention.

[0011] Another advantage of the method of the invention is theversatility for adaptation to any antibody. For example, the methodcould be adapted to detect troponin-I or T by using the antibodyspecific for troponin-I or T attached to a second antibody, such as theantibodies shown herein, that recognizes the detector probe. If highersensitivity is necessary, the polymer probe could be generated to carryhigher numbers of signal compounds. Furthermore, the polymer probe caninclude any kind of signal compound, such as radioisotope, fluorescent,or paramagnetic linked signal compounds.

[0012] All previously existing ELISA radioimmunoassays, dip-stick assaysfor cancer, pregnancy, serum enzymes and probes and any assays utilizingantibodies could be modified according to the method of the invention toprovide enhanced sensitivity. In addition, in vivo application toenhance target signal by using the method of the invention is alsopossible.

[0013] The following examples are presented to illustrate the advantagesof the present invention and to assist one of ordinary skill in makingand using the same. These examples are not intended in any way otherwiseto limit the scope of the disclosure.

EXAMPLE I

[0014] Serum immunoassays for intracardiac contractile proteinsconstitute the mainstay for detection of myocyte necrosis associatedwith various cardiovascular disorders. However, myosin heavy chain (MHC)fragments can be detected by immunoassay only after 48 h from the onsetof chest pain. To enhance immunodetection of MHC, monoclonal antibody(MAb) R11D10 specific for cardiac MHC was covalently linked to MAb4G4-1D5 specific for DTPA. The probe consisted of DTPA-modifiedpolylysine (28:1 molar ratio) covalently linked to horse-radishperoxidase (6 moles/mole polylysine)(PL-DTPA-HRP). Porcine cardiacmyosin (PCM, 1 μg/ml) was used to coat the microtiter wells. Afterovernight incubation and washing, three times, 50 μl each of 5 μg/mlBiMAbor MAb and serial dilutions of PCM (0.001 to 100 μg/ml) or 50 μl ofserial dilutions (1/1 to 1/10000) of patient sera pre-incubated for 1 hat 37° C. were added and incubated for 2 h at 37° C. After washing, thewells were incubated with goat-antimouse IgG-HRP or PL-DTPA-HRP for 2 h.A chromogen, dinitrobenzidine was used to develop the assay. Theaffinity of BiMAb and R11D10 were the same at 1.5×10⁹ L/mole. Thesensitivity of BiMAb was 0.5 ng, whereas that of R11D10 was 0.5 μg (1μg/ml) BiMAb developed with the conventional goat anti-mouse IgG-HRP hada sensitivity of 0.05 μg. Therefore, BiMAb assay has a 1000 foldincrease in sensitivity compared to the conventional immunoassay in thesera of 3 heart transplant patients. Using the BiMAb assay, 2.5, 1.25and 1.3 ng MHC/50 μl serum at 1/10³ dilution, were detected. This BiMAbtechnology can be used in RIA or ELISA by interchanging the HRP probefor radiolabeled probe and should provide more specific in vitrodiagnosis of acute myocardial infarction since detection of MHC is notfeasible at the present time of day 1 of myocardial infarction byconventional immunoassays.

EXAMPLE II

[0015] In a subsequent experiment the DTPA-modified polylysine probe ofExample I was covalently linked to 12 moles of horse-radish peroxidaseper mole of polylysine. The results of the study show that thesensitivity of the bispecific assay of the invention (10⁻⁵ to 100 μg/ml)was at least 10,000 fold better than the conventional immunoassay (0.1μg/ml).

[0016] While the present invention has been described in conjunctionwith a preferred embodiment, one of ordinary skill, after reading theforegoing specification, will be able to effect various changes,substitutions of equivalents, and other alterations to the compositionsand methods set forth herein. It is therefore intended that theprotection granted by Letters Patent hereon be limited only by thedefinitions contained in the appended claims and equivalents thereof.

What is claimed is:
 1. An immunoassay method comprising reacting asample from a patient with a bispecific antibody, said bispecificantibody comprising one antibody specific for a compound to be detectedand a second antibody specific for a compound foreign to said patientsample; and subsequently reacting said sample with a polymer probe, saidpolymer probe comprising a polymer backbone, attached to said polymerbackbone, a compound recognizable by said second antibody in saidbispecific antibody, and at least two detectable signal compoundsfurther attached to said polymer backbone.
 2. The immunoassay method ofclaim 1, wherein said polymer probe comprises at least ten detectablecompounds.
 3. The immunoassay method of claim 1, wherein said detectablesignal in said polymer probe is selected from the group consisting ofradioisotope, fluorescent probe and paramagnetic probe.
 4. Theimmunoassay method of claim 1, wherein said sample from said patient isa blood or serum sample; said bispecific antibody comprises anantimyosin antibody and an antibody against DTPA; and said polymer probeis a polylysine polymer and comprises DTPA and at least 6 HRP as saiddetectable signal compounds.
 5. A bispecific antibody for use in animmunoassay comprising one antibody specific for a compound to bedetected in said immunoassay; and a second antibody specific for acompound foreign to a sample to be assayed in said immunoassay.
 6. Apolymer probe comprising a polymer backbone; attached to said polymerbackbone, a compound recognizable by an antibody in a bispecificantibody; and at least two detectable signal compounds attached to saidpolymer backbone.